PROJECT SUMMARY/ABSTRACT Defense against hypoglycemia is critical for survival, and is of particular importance in the clinically-significant setting of insulin-induced hypoglycemia. Insulin-induced hypoglycemia is prevalent in Type 1 and advanced Type 2 diabetes mellitus, and is associated with a far-ranging negative impact, including reduced work productivity and quality of life, decreased adherence to or recommendations for intensive insulin regimens (resulting in poorer glycemic control), increased incidence of hypoglycemia-related morbidity and unfortunately, occasionally death. Several facets of ghrelin biology suggest that the ghrelin system may participate in the counter-regulatory response to insulin-induced hypoglycemia: Ghrelin secretion is directly stimulated by low glucose. The ensuing raised ghrelin has at its disposal many potential downstream targets with which to influence glucose handling, including stimulation of food intake, reduction of insulin secretion and insulin sensitivity, and enhancement of hepatic gluconeogenesis and hepatic autophagy. Also, the ghrelin system directly interacts with several arms of the traditional counter-regulatory response to hypoglycemia, including stimulation by ghrelin of glucagon and GH secretion, elevation by ghrelin of circulating glucocorticoids and induction of ghrelin release by increased sympathetic nervous system activity. Thus, ghrelin is well-positioned to play a central protective glucoregulatory role during insulin-induced hypoglycemia. However, while ghrelin regulation of blood glucose and by blood glucose have been evaluated in some contexts (e.g. fasting, postprandial, etc.), the overall role of the ghrelin system in the counter-regulatory response to insulin-induced hypoglycemia has not been fully assessed experimentally, for instance by re-creating insulin-induced hypoglycemia in mice without an intact ghrelin system. Here, we will test the concept that ghrelin plays a key, protective, counter-regulatory role in the body's response to insulin-induced hypoglycemia. We will accomplish this by studying effects of insulin-induced hypoglycemia on both ghrelin action and ghrelin secretion using a unique collection of recombinant mouse models. These models include ghrelin-knockout mice and three novel lines: mice lacking insulin receptors exclusively from ghrelin cells, mice lacking GHSRs exclusively from preproglucagon cells, and mice with preproglucagon cell-selective expression of GHSRs on an otherwise GHSR-null background. We will emulate insulin-induced hypoglycemia in these mouse models of altered ghrelin action and secretion using 2 ?gold-standard? paradigms for assessing the counter-regulatory response: insulin-induced (hyperinsulinemic)-hypoglycemic clamp and an insulin bolus-induced hypoglycemia protocol, neither of which have been reported before in mice with altered ghrelin systems. Together with these novel mouse models, our team's experience in studying both ghrelin action and secretion and our expertise with clamp technology and glucose metabolism in mice uniquely position us to address important questions regarding the impact of the ghrelin system on the counterregulatory response to insulin-induced hypoglycemia.